J. Org. Chem. 1981,46, 13-22 Solvent was removed to leave 8 mg of a crude solid, which was purified by column chromatography over 5 g of silica gel, eluting with 60% ether in hexanes, to afford 6 mg (43%) of 6 identical in all respects with material obtained by rearrangement of 2. Bicyclo[5.2.l]decane-4,lO-dione 4-Ethylene Ketal (17). A solution of 0.641 (4.1 mmol) of keto ketal 15 in 15 mL of dry methanol was stirred under dry nitrogen at ice bath temperature with 0.134 g (0.97 mmol) of powdered anhydrous K2C03. A solution of 1.183 g (4.1“01) of dinitrosocarbamate 16 in 20 mL of CHzClzwas added dropwise over 1h and the resulting solution stirred for 3 h, during which time it warmed to room temperature. The reaction mixture was poured into 150 mL of water and 50 mL of brine and extracted with three 50-mL portions of CH2C12. The combined CHzClz extracts were washed with 50 mL of aqueous Na2C03and 50 mL of brine, dried over MgS04, and evaporated to leave 0.884 g of crude product which was chromatographed over 70 g of silica, eluting with hexane-ether (52), to give 0.251 g (34%) of an oil: ‘H NMR (CDC13) 6 1.4-2.5 (14 H), 3.8 (4 H, 9); 13C NMR (CDC13) 6 24.8, 27.1, 33.8, 44.7, 63.2, 64.4; IR (CC14)1735 cm-’; mass spectrum, 210 (0.17), 182 (5.68), 99 (10.59), 55 (11.61); high-resolution mass spectrum calcd for C12H18O3 m / e 210.1255, obsd m / e 210.1249. l-Methylbicyclo[5.2.l]decane-4,lO-dione (18). Method I. A solution of 131 mg (0.74 mmol) of unsaturated bicyclic diketone 7 in 13 mL of 95% ethanol was hydrogenated at atmospheric pressure over 20 mg of 10% Pd on carbon. When 1 equiv of hydrogen had been absorbed, the solution was fdtered to remove the catalyst, and the solvent was removed at reduced pressure to leave 134 mg of crude product which was chromatographed over 8 g of silica, eluting with hexane-ether (21), to afford 95 mg of pure saturated diketone. Method 11. To a solution of 0.13 mL (94 mg, 0.93 mmol) of diisopropylamine in 10 mL of dry DME at ice bath temperature was added dropwise over 15 min 0.7 mL (0.97 mmol) of a 1.38
13
M solution of n-BuLi in hexane. After the mixture was stirred for a further 10 min, 184 mg (0.87 mmol) of ketal ketone 17 in 7 mL of DME was added dropwise over 30 min, and stirring waa continued for an additional 30 min before addition of 1mL (16 mmol) of methyl iodide. After 90 min the reaction mixture was poured into 300 mL of 5% aqueous HC1 before extraction with three 1WmL portions of ether. The combined ether extra& were washed with 100 mL of 5% aqueous HC1 and 100 mL of brine and dried over MgS04. Removal of solvent at reduced pressure left 73 mg of crude product which was chromatographed over 9 g of silica, eluting with hexane-ether (32), to afford 31 mg of the title compound. Both samples gave identical spectral data: ‘H NMR (CDC13) 6 1.05 (3 H, s), 1.5-3.0 (13 H); 13CNMR (CDCld 6 22.1,23.4,29.1, 34.0, 36.2, 38.0, 39.7, 45.3; IR (CC1,) 1740, 1710 cm-’; mass spectrum, 180 (0.75), 162 (0.66), 152 (1.25), 110 (2.51), 97 (5.63), 55 (6.34). Anal. Calcd for C11Hls02: C, 73.30; H, 8.95. Found C, 73.01; H, 8.96.
Acknowledgment. This research was supported by a grant from the National Institutes of Health (CA 12617). Registry NO. 1, 75400-29-2; 2, 75400-30-5; 3, 75400-31-6; 4, 7424-82-0;5,75400-32-7;6,75400-33-8;7,75400-34-9;8,75400-35-0; loa, 75400-36-1; lob, 75400-37-2; lla, 75400-38-3; lla xanthate, 75400-39-4; 12,75400-40-7;13,75400-41-8;14,75400-42-9;15,474697-8; 16, 19935-89-8; 17,75400-43-0; 18,75400-44-1;2-methylcyclopentanone, 1120-72-5;methyl acrylate, 96-33-3; methyl 3-bromo-lmethyl-2-oxocyclopentanepropanoate,75400-45-2; l-methyl-2-0~03-cyclopentenepropanoicacid, 75400-46-3;vinyl bromide, 593-60-2; l~-(benzoyloxy)-~-methyl-1,2,3,3~,4,7,&heptahydro-6(7H)-azulenone ðylene ketal, 75400-47-4; 8a-methyl-3,3a,4,5,&pentahydro2-(trimethylsilyloxy)-1,6(2H,7H)-azulenedione6-ethylene ketal, 75400-48-5.
Reactions of Steroid Salts with Hexachlorocyclotriphosphazene’ Harry R. Allcock,* Timothy J. Fuller, and Kiyotoshi Matsumura Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 Received May 29, 1980
The sodium salts of the steroids desoxoestrone (VI),estrone (VII),8-estradiol (VIII),17a-ethynylestradiol (IX),estradiol 3-methyl ether (XIV), and 1,4-dihydroestradiol 3-methyl ether (XV)were found to react with hexachlorocyclotriphosphazene(111) to yield species of the general formula N3P3C16(OR)(IV). The remaining halogen atoms were replaced by methylamine to yield hydrolytically stable derivatives of formula N3P3(NHCH,),(OR) (V).These are model compounds for the analogous steroid-substituted phosphazene linear high polymers. Aliphatic steroids such as cholesterol (X), dihydrocholesterol (XI), pregnenolone (XII),estradiol 3-methyl ether (XIV), or 1,4-dihydroestradio13-methyl ether (XV)underwent dehydration as well as substitution in contact with 111. Mestranol (XVII)as its 17-sodium salt did not react rapidly with 111, presumably for steric reasons. At elevated temperatures, estrone 3-methyl ether (XVI)interacted with I11 by a complex process that involved elimination of hydrogen chloride. All the chlorine atoms in I11 could be replaced by a reaction with the sodium aryloxide salt of estrone (VII). The replacement followed a nongeminal pathway. The significance of these results for the synthesis of the related high polymers is discussed.
Considerable interest exists in the synthesis of high molecular weight polymers that may function as carrier molecules for chemotherapeutic Phosphazene (1)For a previous paper in this series, see P. P. Greigger and H. R. Allcock, J. Am. Chem. Soc., 101,606 (1979). (2) L. G. Donaruma in “Synthetic Biologically Active Polymers”, A. D. Jenkins, Ed., Pergamon Press, New York, 1975, Chapter 1. (3) H.-G. Batz, Adv. Polym. Sci., 26, 25 (1977). (4) L. G. Donaruma and 0. Vogl, Ed., “Polymeric Drugs”, Academic Press, New York, 1978. (5) A. Zaffaroni and P. Bonsen, ref 4, p 1. (6) H. R. Allcock, R. W. Allen, and J. P. OBrien, J. Chem. Soc., Chem. Commun., 717 (1976).
high polymers with steroidal side groups could be potentially useful drugs or prodrugs, assuming that a slow hydrolytic release of the steroid could occur in vivo. Moreover, the possibility exists that steroidal phosphates might be released during hydrolysis, species that have themselves been used in steroid therapy.*” However, effective (7) R. W. Allen, J. P. OBrien, and H. R. Allcock, J. Am. Chem. SOC., 99, 3984 (1977). (8) H. R. Allcock, R. W. Allen, and J. P. O’Brien, J. Am. Chem. SOC., 99, 3987 (1977). (9) R. J. W. Cremlyn and I. Khattak, Phosphorus, 6,237 (1976). (10) R. B. Brownfield and W. Schultz, Steroids, 2, 597 (1963).
0022-3263/81/1946-0013$01.00/00 1981 American Chemical Society
14
J. Org. Chem., Vol. 46, No. 1, 1981
Allcock, Fuller, and Matsumura Chart I
Vli
VI
IX
XI
X
XI I
XI1 I
#OH
&OH
Me0
Me 0
xv
XIV
macromolecular syntheses and reactivity studies usually require prior studies with small-molecule analogues. Cyclic trimeric phosphazenes are useful models for the linear high polymers,14and in this paper we discuss a series of model compound studies with steroidal-substituted cyclic trimeric phosphazenes. The compounds studied in this work have the general structure shown in I, where OR is a steroidal residue bound
I1
hexachlorocyclotriphosphazene,(NPC12)3,with alkoxides and aryl oxides are normally rapid and complete. However, evidence exist^'^^^^ that bulky nucleophiles generate steric hindrance effects, both with respect to the introduction of the nucleophile itself and to the subsequent introduction of other side groups. Nucleophiles based on a steroid framework offer a means for an exploration of such steric effects. The specific reaction sequence discussed in this paper is the conversion of 111 to IV and V. The alternative
I11
to a phosphazene skeleton via the 3- or 17-position. These species serve as models for high polymers of structure 11. In principle, the group X can be a cosubstituent unit designed to solubilize the molecule in water (NHCH3) or destabilize it to hydrolytic attack (NHCH2COOC2HS,15 imidazolyl,16 or NH2”). In this paper, we restrict our attention exclusively to the use of the methylamino group as a cosubstituent species, although it is recognized that the use of amino acid ester or imidazolyl groups could provide hydrolysis products which are more biocompatible. From a fundamental point of view, the attachment of steroidal residues to a phosphazene ring offers the prospect of mechanistic comparisons with the less complex alkoxyand (ary1oxy)phosphazenes that have already been investigated in detail.l* For example, the reactions of ~~
~
(11)The phosphate group has also been used to facilitate the administration of aqueous solutions of corticosteroid esters without reduction of physiological activity?J2J3 (12)T. Reichstein and W. Schlindler,Helu. Chim. Acta, 23,669 (1940). (13)S.Harai, R. G . Harvey, and E. V. Jensen, Tetrahedron, 22,1625 i1SF;fi) ~____,_
(14)H. R.Allcock, Acc. Chem. Res., 12,351 (1979). (15)H. R. Allcock, T . J. Fuller, D. P. Mack, K. Mataumura, and K. M. Smeltz, Macromolecules, 10, 824 (1977). (16)H.R. Allcock, T. J. Fuller, K. Mataumura, and D.Mantz, unpublished work. (17)D.B. Sowerby and L. F. Audrieth, Chem. Ber., 94, 2670 (1961). (18)H. R. Allcock, “Phosphorus-Nitrogen Compounds”. Academic Press, New York, 1972.
IV
V
sequence-an introduction of the methylamino residues first, followed by the steroid residue-was not attempted, because of the known high reactivity of methylamine toward 111 and the probability of complete chlorine replacement by the amine. The principal steroids used in this work were desoxoestrone (VI), estrone (VII), 170-estradiol (VIII), 17aethynylestradiol (IX), and estradiol 3-methyl ether (XIV) (see Chart I). However, a number of other steroids were also examined in an attempt to explore the scope of these reactions. These included cholesterol (X), dihydrocholesterol (XI), pregnenolone (XII), 20-0xo-5a-pregnan-3/3-01 (XIII), 1,4-dihydroestradiol3-methylether (XV), estrone 3-methyl ether (XVI), and mestranol (XVII). These steroids were chosen for the following reasons. The steroidal residue from VI (itself derived from VII) represented the simplest steroid structure that extended (19)H. Schadow and H. Scheler, 2. Chem., 13, 223 (1973). (20)H. R.Allcock, S. D. Wright, and K. M. Kosydar, Macromolecules, 11, 357 (1978).
Hexachlorocyciotriphosphazene
the known reactions of phenoxide ion18with 111. As such, it formed a primary model against which the other steroid structures could be compared. The structures derived from VI1 and XI1 provided a test for the stability of keto or acetyl functions under the chosen reaction conditions. Furthermore, VII-IX, XII, and XI11 should be pharmacologically active in the covalently bound form (V). Compounds X-XI11 also constituted examples of aliphatic 3-hydroxy functional groups that could be compared with the known reactions of simple alkoxides with 111. Steroid XIV was used to explore the reactivity of a secondary alcohol function at the 17-position and to monitor the role played by the angular 18-methyl group. Steroid XVII was utilized both as an example of a species with a tertiary alcohol function at the 17-position and because it is active as a birth control agent. Compound XVII or XIV was used as a control to model the reactivities (a) of the l7-position, (b) of aliphatic alcohols, and (c) of VIII or M, respectively. Cholesterol (X) provided a control for monitoring the stability of the acetyl group at position 20 in XI1 or XI11 under the reaction conditions employed. Species XI or XI11 was employed as a control to deduce the behavior of the double bond in X or XII. Three of the steroidsdesoxoestrone (VI), cholesterol (X), and estradiol 3-methyl ether (X1V)-were chosen because they possess only one reactive group and, thus, were not expected to participate in ring-coupling reactions. The C-3 methyl ether groups of the steroids XIV-XVII were expected to block reactions at these sites. Compounds VI-M offered the prospect that their reactions could be monitored by electronic spectroscopy. Model nucleophiles for steroids VI-IX were provided by phenol and sodium phenoxide and for VII, XII, and XI11 by cyclohexanone or XVI and their sodium salts. With these facts in mind, this study was carried out in an attempt to answer the following questions. (1) Can bulky steroid molecules be attached through the 3- or 17-hydroxy positions to a cyclophosphazene ring to yield species such as IV or V, and what are the preferred experimental conditions needed to achieve that end? (2) Does the introduction of the steroidal residue sterically inhibit the introduction of a second substituent group such as methylamino? (3) Are the steroidal residues in species such as IV stable to subsequent reactions needed to introduce methylamino residues, and is the phosphazene skeleton stable to reactions required to remove protecting groups from the steroid? (4) Might free steroids such as X-XV or XVII undergo dehydration rather than substitution in the presence of (NPC12)3?21 (5) Can phosphazenes derived from X-XI11 undergo a migration of alkyl groups from oxygen to skeletal nitrogen in a manner reminiscent of the known behavior of simple alkoxycyclophosphazene?22
Results and Discussion Formation of Steroid Salts. The steroids were converted to their sodium salts by treatment with sodium hydride either at room temperature or in boiling tetrahydrofuran (THF). Because steroids, such as VII-IX, XII, XIII, and XVII, contain additional active groups that could react with sodium hydride, considerable care was taken to ensure that the reaction of the hydroxyl group was the only process that occurred. For example, estrone (VII) was allowed to (21)M.Kajiwma and H. Saito, Kogyo Kagaku Zasshi, 74,619(1971); Chem. Abstr., 75,34923 (1971). (22)B. W.Fitzsimmons, C. Hewlett, and R. A. Shaw, J. Chem. SOC., 4459 (1964).
J. Org. Chem., Vol. 46, No. 1, 1981 15 react with sodium hydride under mild conditions (see Experimental Section) in very dilute solution to discourage self-condensation reactions. The 17-carbonyl group was virtually unaffected under these conditions. A more detailed study entailed the interaction of XVI with sodium hydride under more forcing conditions, followed by exposure to (NPC1J3. No detectable quantity of phosphazene enol was identified, and essentially all of XVI was recovered unchanged. The ease of sodium salt formation during the reaction of the steroids VI-XVII with sodium hydride was dependent on the relative acidities of the alcohols. Aromatic (3-position) alcohols such as VI-IX reacted immediately with sodium hydride at 25 "C. However, no detectable reactions between the steroids X-XIV, XVI, or XVII with sodium hydride and I11 were evident (with the use of 31P NMR analysis) even after 24 h at 25 "C. Therefore, it was possible to generate the monosodium salts selectively at the C-3 positions of the steroids VII-IX (at 25 "C) without affecting the other functional sites. Steroids such as X and XIV reacted with sodium hydride after prolonged boiling in THF (>12 h). It was even possible to prepare the salts of the steroids X-XIV with sodium hydride in the presence of I11 at 25 "C but at a much slower rate than at the higher temperature. The C-17 alcohol and C-20 alcohol or keto groups of the steroids VII-IX and XII-XVII were unreactive with I11 even after 24 h in the presence of sodium hydride (see Experimental Section). However, the sodium salts of the C-3 aromatic alcohols of VI-IX reacted rapidly under these same reaction conditions. Thus, multifunctional steroids such as VII-IX reacted with 111 selectively at C-3 to form IV without the w e of protecting groups. It should be noted that these reactions were carried out with dilute solutions of the reagents, usually at 25 "C. In concentrated solutions, and especially at higher temperatures (-70 "C), side reactions became serious (see the following sections). Two different procedures were followed to generate salts at the 17-position of mestranol (XVII) without reaction of the acetylenic unit. First, reaction of XVII with excess sodium hydride, followed by removal of the excess sodium hydride, and addition of a stoichiometric equivalent of XVII to the reaction mixture ensured the absence of the acetylide salt. Second, a quantitative addition of methyllithium to XVII yielded the lithium alcoholate. Reactions of Steroid Salts with (NPCl2)3. General Features. The reactivities of the alcohol groups or sodium salts of steroids VI-XVII with (NPC12)3were monitored by 31PNMR methods. Treatment of excess I11 with the sodium salts of VI-IX or XIV in dilute solutions resulted in the rapid formation of monosteroidal-substituted phosphazenes of type IV. Sodium chloride precipitated. The more acidic steroidal alcohols (such as VI-IX) gave higher yields of IV,a result that was ascribed to the relative efficiency of the steroidal salt formation rather than to a direct steric influence during (ary1oxy)phosphazene formation. In contrast to the behavior of the sodium salts, the reactions of the alcohol groups of steroids VI-XVII with I11 were a very slow processes and required long reaction times before a detectable formation of IV resulted. No products of type IV or V were isolated when the steroidal reagent was derived from XVI or XVII, and only low yields of IV were detected with X-XIII. Because of their hydrolytic instability, the products of structure IV derived from desoxoestrone (VI), estrone (VII), @-estradiol(VIII), a-ethynylestradiol (IX), cholesterol (X), dihydrocholesterol (XI), and estradiol 3-methyl
16
Allcock, Fuller, and Matsumura
J. Org. Chem., Vol. 46, No. 1, 1981 Scheme I
1 ' ether (XIV) were not isolated in an ultrapure state. They were characterized in solution by 31Pand 13C NMR, infrared, or electronic spectral techniques and were used directly for conversion to V. Reaction of Estrone with 111. Estrone (VII; as its sodium aryloxide salt) reacted with I11 by a nongeminal substitution pathway to form species XIX-XXVII (Chart 11). The low solubility of the sodium salt of VI1 and its steric bulk affected the rate and the number of estrone groups attached to 111. Compounds XIX-XXI were formed at 25 OC (within 12 h), whereas the formations of XXII-XXV required longer reaction times (24 h) under the same experimental conditions. Species XXVI and XXVII were generated only with the use of more forcing conditions. It is surprising that isomers XXII and XXIII were formed in equal amounts. Similarly, the yields of isomers XXIV and XXV were approximately the same. Although the presence of a steroid residue on I11 induces a nongeminal substitution pathway, it has no apparent influence on the stereochemistry of the steroid attachement to the other phosphorus atoms. Special Case of Pregnenolone (XII) and Cholesterol (X). Very low yields ( residue from XIV (14.2) = OCH2CF3(14.1) > residue from X (13.1) = residue from XI (12.4) > residues from VI-IX (11.7-12.0) > O c a 5 (9.9). This parallels the expected order of increasing electron supply from the side group to phosphorus in the manner seen for other cyclophosphazene derivatives.% The replacement of chlorine in IV by methylamino to give V markedly reduced the chemical shift difference between the A and B2 phosphorus atoms and brought about an overlap of the two. 13C NMR spectra were used to confirm the retention of the steroidal framework following the linkage and co-substitution reactions. Although the 13C NMR spectra were not sufficiently resolved to permit the identification of every carbon nucleusm in the steroid structure for N3P3(0R)C15(IV) or N3P3(OR)(NMeH), (V) (where HOR = VI, VII, and XIV),no change in the absorbances of the aromatic nuclei for steroid XIV occurred as a result of the interaction with sodium hydride or with (NPCl& This indicated that no major side reactions had occurred that involved the ether function at the 3-position of XIV. For steroid XIV, only a slight change in the relative intensity and no change nucleus in the chemical shift at 82 ppm was observed for the
'w
22
J . Org.Chem. 1981, 46, 22-27
at the 17-position after attachment to the phosphazene ring. By contrast, changes in the chemical shifts for the resonances of aromatic '3c nuclei for steroid VI were detected following reaction of the sodium salt of VI with (NPCl& 13CNMR spectroscopy (together with mass spectrometry) was also used to confirm the identity of the unreacted steroids VI, VII,and XIV isolated from the reaction products. This confirmed the absence of side reactions. No mass spectral or 13CNMR spectral changes occurred after XVII had been treated with liquid methylamine (for 72 h).
Acknowledgment. This work was supported by the Public Health Service through Grant No. 5ROlHL1141809. Fb&tv NO.111,940-71-6; IV (HOR = VI), 74026-89-4;IV (HOR = VII),75267-32-2; IV (HOR = VIII), 75267-33-3; IV (HOR XIV), V (HOR = VI),74026-90-7;V (HOR = VII), 74026-91-8; 75267-34-4;
V (HOR = VIII), 74026-95-2;V (HOR = IX),75267-35-5; V (HOR = X), 74026-93-0; V (HOR = XIV),75283-90-8;V (HOR = XV),
75267-37-7; VI, 53-63-4;VI d u m salt, 74026-883;VII, 53-16-7;VI1 sodium salt, 74040-94-1; VIII, 50-28-2; VI11 sodium salt, 17181-19-0; IX,57-63-6;IX sodium salt, 75267-38-8;X,57-88-5; XI,17608-41-2; XI1 sodium salt, 75267-39-9;XI11 sodium d t , 75267-40-2;XIV, 1035-77-4; XIV sodium salt, 74026-92-9; XVI,1624-62-0;XVII,7233-3;X M (HOR IX),75267-41-3;XIX (HOR = X),75267-42-4; XIX (HOR = XI),75267-43-5; XX (HOR = VII), 75267-44-6;XXI (HOR = VII), 75330-94-8; XXII (HOR VII), 75299-11-5;XXIII (HOR = VII), 75363-53-0;XXIV (HOR = VII), 75283-91-9;X X V (HOR = VII), 75331-70-3; XXVI (HOR = VII), 75267-45-7;XXVII (HOR = VII), 75267-46-8;N3PJ&(OCH&F3), 13053-90-2;NsPaClS(OC&,),302810-2;N$3Cb(OC$14Br-p), 3887-16-9;NSP3C1,(0H), 75267-47-9;N3P3C16(ONa),75283-92-0;N3P3Cl,(OCH2CF,)z, 75267-48-0; N3P3C14(OH)z, 75267-49-1; N$3Cl,(ONa)2, 75267-50-4; N3P&13(OCH&F&, 75330-95-9;NSP3C12(OCHzCF3)4, 75267-51-5; N$&l(OCHzCFJ6,55975-53-6; N$&l(OC&)6, 5032-39-3;[NE'(@ CHzCF3)~]3,1065-05-0;[NE'(OC&)2]3, 1184-10-7; [NP(NHCHa)2]3, 1635-63-8; NsP3(NHCH&(OCH&F3), 75283-93-1; NSP3(NHCHS)S(OH), 75267-52-6;N3P3(NHCHJ6(ONa), 75267-53-7;N3P3(NHCH&(OH), 75267-54-8; N3P3(NHCH3)4(0Na)2, 75267-55-9.
Ichthyotoxic and Cytotoxic Metabolites of the Tropical Brown Alga Stypopodium zonale (Lamouroux) Papenfuss' William H. Gerwick and William Fenical* Institute of Marine Resources, Scripps Institution of Oceanography, La Jolla, California 92093 Received May 12, 1980
Seven new natural products, stypoldione, stypotriol, stypodiol, epistypodiol, epitaondiol, 2-(geranylgeranyl)-5-methyl-l,4-benzohydroquinone, and 2-(geranylgeranyl)-5-methyl-1,4-benmquinone, and two previously reported metabolites, taondiol and atomaric acid, have been isolated from the Caribbean brown alga Stypopodium zonale. Taondiol was obtained as the enantiomer of that previously reported. The structures of the new compounds were based upon the previously described X-ray structure of stypoldione and upon detailed 13Cand 'H NMR analyses. Nearly all the compounds isolated showed toxic or strong narcotic effecta upon the reef-dwelling fiih, Eupomacentrus leucostictus, and stypoldione was found to be an extremely potent inhibitor of synchronous cell division in a fertilized sea urchin egg assay.
In the course of our investigations of the chemical defense strategies of tropical marine algae, we are continuing to explore the natural products chemistry and subsequent chemical ecology of brown seaweeds of the family Dictyotaceae (Dictyotales). One such example in this family is the brown alga Stypopodium zonule (Lamouroux)Papenfuss, which is found to grow luxuriantly in the Western Caribbean Sea where herbivorous invertebrates and fishes are particularly abundant.2 In an earlier communication, we reported the structures of two ichthyotoxic metabolites, stypoldione and stypotriol, isolated from S. ona ale.^ In this complete report of our investigations of this alga, we describe the structures of several new biologically active compounds and amplify upon our initial findings. Stypopodium zonale was initially chosen for study based on observations that when the freshly collected plant was placed in a well-aerated aquarium, the alga was found to excrete rust-colored substances which proved to be toxic to the Caribbean herbivorous fish Eupomacentrus Zeucostictus. During the course of 1week, with daily changes of the aquarium water, the plants continued to excrete ichthyotoxic substances with no visible degeneration of the plants themselves. Diethyl ether extraction of the water (1)Inshore Marine Shallow Water Ecosystem Project Contribution no. 60. (2) Ogden, J. D.; Lobel, P. S. Enuiron. Biol. Fishes 1978,3(1), 49. (3) Gerwick, W. H.; Fenical, W.; Fritsch, N.; Clardy, J. Tetrahedron Lett. 1979,145.
yielded a complex mixture of UV-absorbing compounds, the major component of which was the bright red oquinone, stypoldione (1). Stypoldione in seawater, at 1.0 pg/mL, induced the same toxic symptoms as the algaetreated water. The chloroform-methanol extract of the fresh alga proved even more toxic than pure 1, suggesting that a more potent intracellular toxin was present. Indeed, when algal extracts were rapidly chromatographed in the field, a white solid, stypotriol (2), was obtained, which rapidly began turning pink and then red. The hydroquinone 2 was substantially more toxic than 1 (LD N 0.2 pg/mL), but as indicated by the color change, it is rapidly air oxidized to stypoldione such that precise concentrations in seawater cannot be p r o d u ~ e d . Hence, ~ the major toxic metabolite of S. zonale is the o-hydroquinone 2, which when excreted, is rapidly air oxidized to the o-quinone 1. The structures of 1 and 2 were communicated earlier3based upon a conclusive X-ray analysis of 1. Complete analysis of the extract has now resulted in the isolation of compounds 2 and 4-10 (Chart I), and several of these compounds contribute to the observed overall ichthyotoxicity of 5'. zonale. Contrary to our earlier report, the alga does contain taondiol (9): as well as atomaric acid however, the (4) The details of this fish toxicity bioassay were recently s u m m d , see: Sun,H. H.; Fenical, W. Tetrahedron Lett. 1979,686. ( 5 ) G o n d e z , A. G.; Darias, J.; Martin, J. D. Tetrahedron Lett. 1971, 2729.
0022-3263/81/1946-0022$01.OO/00 1981 American Chemical Society
